3.3. Triparental Mating of P. aeruginosa

To prepare a P. aeruginosa strain which is able to produce GFP, triparental mating was carried out. For this conjugation of 3 bacteria, E. coli Nissle pVLT31-eGFP was used as a donor strain, P. aeruginosa PAO1 was used as an acceptor strain, and E. coli DH5α pRK2013 was used as a carrier strain. First, one preculture of each strain was prepared by inoculating 5 mL LB medium (Carl Roth GmbH + Co. KG) with a single colony. After that, the optical density at 600 nm was measured with the VWR^® Spectrophotometer UV-1600PC, and 1 mL of bacteria suspension with OD₆₀₀ = 0.6 was pipetted successively in one 1.5-milliliter reaction tube. For this, the bacterial suspension was centrifuged for 2 min at 11,000× g and the supernatant was removed. Then, the bacteria mix was resuspended in the last drop of the LB medium supernatant, pipetted on a prepared LB medium agar plate, and incubated for 3 h at 37 °C. After that, the bacteria colony was resuspended in 5 mL of LB medium with 50 µg/mL tetracycline (Carl Roth GmbH + Co. KG) and 100 µg/mL ampicillin (Carl Roth GmbH + Co. KG), then diluted to 10⁵–10⁶ to gain 10³ bacteria/mL and plated on agar plates with 50 µg/mL tetracycline and 100 µg/mL ampicillin. Additionally, an undiluted bacteria colony was plated on one new agar plate. Then, the plates were incubated at 37 °C for 16 h. After this, the grown P. aeruginosa PAO1 pVLT31-eGFP colonies were plated on a new agar plate with 50 µg/mL tetracycline and 100 µg/mL ampicillin to separate the colonies. After a renewed incubation for 16 h at 37 °C, these P. aeruginosa PAO1 pVLT31-eGFP bacteria colonies were used for the next experiments.

For bacteria cultivation, a preculture for each bacterial strain was prepared. Therefore, 5 mL of LB medium was transferred into a test tube sterilized by autoclaving. For the bacteria P. aeruginosa PAO1 pVLT31-eGFP and E. coli Nissle pVLT31-eGFP, 10 µg/mL tetracycline was additionally added. To the control strain P. aeruginosa PAO1, no tetracycline was added. After adding the cells to the medium via inoculation loop, the precultures were incubated for 16 h at 37 °C with shaking at 150 rpm. In the next step, the OD₆₀₀ values of the precultures were measured; then, 25 mL LB medium supplemented with 10 µg/mL tetracycline was inoculated to a starting OD₆₀₀ of 0.05. No tetracycline was added to P. aeruginosa POA1 cultures without the plasmid pVLT31-eGFP. The cultivation was carried out at 37 °C with shaking at 150 rpm. After a cultivation time of 30 min and 1 to 7 h every hour, the OD₆₀₀ was monitored. At OD₆₀₀ = 0.6, the bacteria cultures of P. aeruginosa PAO1 pVLT31-eGFP and E. coli Nissle pVLT31-eGFP were induced with 0.4 mM Isopropyl-ß-d-1-thiogalactopyranoside (IPTG, Carl Roth GmbH + Co. KG). After induction, the cultivation was continued for another 3 h until the stationary phase of the bacteria was reached, and then the cells were counted under a microscope and a Thoma cell counting chamber. Then, the bacterial suspensions were diluted to 12,500 cells/250 µL for the following experiments.